DNA sequencing

Nucleic Acid Chemistry

Third term 2022-2023

Randy Bryant

Department of Biochemistry and Molecular Biology

Johns Hopkins Bloomberg School of Public Health

DNA polymerases Catalyze the synthesis of DNA chains

3´ 3´

5´

dNTP

Biochemistry, 6 th ed. Freeman 2007

Use deoxynucleoside triphosphates (dNTPs) as precursors

Extend DNA chains by adding one nucleotide unit at a time to

the 3´-end of a primer strand
 Template-directed DNA polymerization

primer

template Biochemistry, 6th ed. Freeman 2007

DNA polymerase uses single-stranded DNA as a template

Selects and adds the nucleotide that is complementary to the
corresponding nucleotide in the template strand
Dideoxy DNA sequencing
Fred Sanger and dideoxy DNA sequencing

Fred Sanger
 Dideoxy chain termination

deoxyribonucleoside dideoxyribonucleoside
triphosphate triphosphate
(dNTP) (ddNTP)

extends DNA synthesis terminates DNA synthesis

 Dideoxy chain termination

3´
H

ddNTP
3´
H H

no -OH here

Cannot add another nucleotide when a ddNTP has been incorporated

into a growing DNA chain
Dideoxy chain terminators

ddATP ddGTP

ddCTP ddTTP
 Dideoxy sequencing
Convert DNA to be sequenced into a single-stranded form
Anneal short primer adjacent to region to be sequenced
Carry out primer extension reaction using DNA polymerase
with:
High concentration of the four normal dNTPs
and
Low concentration of one ddNTP (1:100)

Under these conditions:

The chance of incorporating a ddNTP in place of a dNTP will
be small – so some DNA synthesis will occur

But a ddNTP will be incorporated at some point during the

reaction and terminate DNA synthesis
Example: If the reaction was carried out with four normal dNTPs and
ddATP:

5´ 3´

primer GACT
template CTGATGGATCGATCGAGC

primer GACTA 5 nt
template CTGATGGATCGATCGAGC

primer GACTACCTA 9 nt
template CTGATGGATCGATCGAGC

primer GACTACCTAGCTA 13 nt
template CTGATGGATCGATCGAGC

Would generate a mixture of fragments corresponding to termination at

each position where there was a T in the template
Example: If the reaction was carried out with four normal dNTPs and
ddTTP:

5´ 3´

primer GACT
template CTGATGGATCGATCGAGC

primer GACTACCT 8 nt
template CTGATGGATCGATCGAGC

primer GACTACCTAGCT 12 nt
template CTGATGGATCGATCGAGC

primer GACTACCTAGCTAGCT 16 nt
template CTGATGGATCGATCGAGC

Would generate a different mixture of fragments corresponding to

termination at each position where there was a A in the template
 Dideoxy DNA sequencing

Molecular Cell Biology, Freeman 2008

Carry out four parallel reactions using the four normal dNTPs and each
of the four ddNTPs
Analyze the products of each individual reaction by polyacrylamide
gel electrophoresis
 DNA sequencing gel

longer Separates DNA strands

differing in length by a
single nucleotide

Termination products
appear as a ladder
shorter of bands

primer

Can read the sequence of the synthesized strand directly off the gel
Will be complementary to the sequence of the template strand being
analyzed
 Dideoxy DNA sequencing

Fred Sanger DNA sequencing gel

Sanger et. al. “DNA sequencing with chain-terminating
inhibitors” PNAS 74, 5463 (1977)

Can sequence ~300-500 bases per reaction

 Sanger dideoxy DNA sequencing

Sanger used Klenow polymerase in his original paper

But:

Is a relatively slow polymerase: 30-45 nucleotides/sec

Is not very processive: 10-50 nucleotides/binding event

The 3´-5´ exonuclease tends to degrade primer strand

and interfere with efficient primer extension

So Klenow polymerase is not an optimal polymerase

for DNA sequencing
 T7 DNA polymerase

Has higher rate of polymerization: 300 nucleotides/sec

Has higher processivity: > 1000 nucleotides/binding event
(with thioredoxin subunit)

Has no 5´-3´ exonuclease activity

So is a more efficient polymerase than Klenow

But the 3´-5´ exonuclease activity still limits the efficiency of

the reaction for DNA sequencing
 SequenaseTM

Chemically modified version of T7 DNA polymerase

Treatment with oxidizing agent inactivates the

3´-5´ exonuclease activity (0.01%)

But retains normal polymerase activity

So was useful for dideoxy sequencing

First introduced in 1987 and was sold commercially

 SequenaseTM version 2.0

Modified form of T7 DNA polymerase

Has a 28 amino acid deletion that removes the

3´-5´ exonuclease domain

So does not degrade primers

Polymerase activity is not altered by the deletion of the

3´-5´ exonuclease domain

Incorporates dideoxy-NTPs and other nucleotide analogs

Sold commercially: Used for dideoxy DNA sequencing

Automated DNA sequencing
DNA sequencing with fluorescent chain terminators
 Fluorescently-labeled dideoxy chain terminators

*
*

TT CC

* *

AA GG

Each of the fluorophores has a distinctive emission maximum and can be

distinguished by fluorescence spectroscopy
Prober et. al. “A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides” Science 238, 336 (1987)
Demonstration of the fluorescently-labeled dideoxy terminator method

normal labeled

Worked with Sequenase, but not

with Klenow polymerase

Prober et. al. “A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides” Science 238, 336 (1987)
Demonstration of the fluorescently-labeled dideoxy terminator method

Prober et. al. “A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides” Science 238, 336 (1987)
 Automated dideoxy DNA sequencing

Uses the four ddNTPs, each labeled with

a different fluorescent group (color)

Termination products are separated

by capillary gel electrophoresis

The colors of the different termination products are determined using

a fluorescence detector as they elute from the gel

Produces a DNA sequence trace that can be converted into a DNA

sequence
Can sequence up to 700-900 bases per reaction
 Automated dideoxy DNA sequencing

Applied Biosystems DNA sequencer DNA sequence trace

Uses a modified form of Taq I DNA polymerase

dNTP/ddNTP selectivity

Compared the dNTP/ddNTP selectivity

of:

E. coli DNA polymerase I

Taq I DNA polymerase

T7 DNA polymerase
 dNTP/ddNTP selectivity

Primer extension reactions were carried

out with a 6:1 ratio of dNTPs/ddNTPs:

G, A, T, C

T7 polymerase incorporates ddNTPs

more efficiently than do Pol I and
Taq I polymerase

Tabor and Richardson, “A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy-and
dideoxyribonucleotides” Proc. Natl. Acad. Sci. USA 92, 6339 (1995)
 Basis for dNTP/ddNTP selectivity

Pol I polymerase: Phe762

Taq I polymerase: Phe667

T7 polymerase: Tyr526

dNTP binding site (Pol I)

Hypothesized that a hydroxyl group at either the 3´-position of the

substrate (dNTP) or in the active site (Tyr) was required to stabilize
a catalytic Mg2+ ion
Tabor and Richardson, “A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy-and
dideoxyribonucleotides” Proc. Natl. Acad. Sci. USA 92, 6339 (1995)
 dNTP/ddNTP selectivity

Replacing Tyr526 of T7 polymerase with

Phe decreased ddNTP incorporation
and
Replacing Phe762 of Pol I, or Phe667 of
Taq I, with Tyr increased ddNTP
incorporation

Were able to re-engineer the dNTP/ddNTP

specificity of these DNA polymerases

Tabor and Richardson, “A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy-and
dideoxyribonucleotides” Proc. Natl. Acad. Sci. USA 92, 6339 (1995)
 Automated dideoxy DNA sequencing

Applied Biosystems DNA sequencer DNA sequence trace

Uses a modified form of Taq I DNA polymerase (F667Y)

Human genome sequencing project

Automated dideoxy sequencing was used

for the first human genome sequencing
project (2001)

Cost ~$100,000,000

Was a composite sequence derived from

the DNA from a number of individuals
DNA pyrosequencing
DNA sequencing by pyrophosphate release
 Pyrosequencing reaction steps

DNA polymerase
(NA)n + nucleotide (NA)n+1 + PPi

ATP sulfurylase
PPi + APS ATP + SO42-

luciferase
ATP + luciferin + O2 AMP + PPi + oxyluciferin

+ CO2 + light
1. DNA polymerase

Each nucleotide incorporation step is accompanied by the

release of one pyrophosphate
2. ATP sulfurylase

- - - - - -
+ +
-
-

- - -

-
- -

pyrophosphate adenosine adenosine sulfate

5´-phosphosulfate 5´-triphosphate
(APS) (ATP)

Each pyrophosphate is used to produce one ATP

3. Luciferase

Each ATP is used to convert one molecule of luciferin to oxyluciferin

and is accompanied by the emission of light
 Pyrosequencing reaction steps

DNA polymerase
+ dNTP

+ APS/ATP sulfurylase

/luciferase

Is a sequencing by synthesis method

Uses normal dNTPs

 Original pyrosequencing reaction scheme

The four different dNTPs were added

template
primer
sequentially to a primer/template
DNA
polymerase
Nucleotide incorporation was detected
by the sulfurylase and luciferase
reactions - as a flash of light

Signal was proportional to the number

of nucleotides that were incorporated

Unincorporated dNTPs were washed

template
extended primer
away and cycle was repeated

Enzymatic Luminometric Inorganic pyrophosphate Detection Assay

Ronaghi et. al. “Real-time DNA sequencing using detection of pyrophosphate release” Anal. Biochem. 242, 84 (1996)
Demonstration of the pyrosequencing method

primer
template

dNTP addition
sequence

This experiment was carried out with Klenow (exo-) polymerase

Also tried Klenow polymerase and Sequenase 2.0

Ronaghi et. al. “Real-time DNA sequencing using detection of pyrophosphate release” Anal. Biochem. 242, 84 (1996)
 Automated DNA pyrosequencing

Qiagen DNA pyrosequencer DNA pyrogram

Uses Klenow DNA polymerase

454 DNA sequencing
 454 DNA sequencing

The first next-generation DNA sequencing technology

Developed in 2008

Features the use of a solid-phase version of the

pyrosequencing method

Adapted so that many sequencing reactions could

be carried out in parallel

Used for genomic sequencing

 454 sequencing

Genomic DNA is fragmented

DNA fragments are ligated to

adapters which contain specific
PCR primer binding sequences

Ligated fragments are then

separated into single strands

Rothberg and Leamon, “The development and impact of 454 sequencing” , Nature Biotech. 26, 1117 (2008)
 454 sequencing

Fragments are annealed to primers

that are attached to beads - one
fragment per bead
primer
Individual beads are isolated in
DNA
the droplets of a PCR reaction
mixture/oil emulsion

PCR amplification PCR amplification occurs within

One strand of the amplified DNA will
be connected to the primer attached each droplet (microreactor)
to the bead

Clonal DNA amplification

Rothberg and Leamon, “The development and impact of 454 sequencing” , Nature Biotech. 26, 1117 (2008)
 454 sequencing

Emulsion is broken

DNA strands are denatured

Beads carrying the covalently

attached single-stranded DNA are
deposited into wells of a fiber-
optic slide
One bead per well

Rothberg and Leamon, “The development and impact of 454 sequencing” , Nature Biotech. 26, 1117 (2008)
 454 sequencing

Smaller beads containing

immobilized pyrosequencing
enzymes are added to each
well:

ATP sulfurylase and luciferase are covalently attached to the smaller beads

Rothberg and Leamon, “The development and impact of 454 sequencing” , Nature Biotech. 26, 1117 (2008)
 454 sequencing

Add: Sequencing primer

DNA polymerase
Four dNTPs

Pyrosequencing reactions begin

Rothberg and Leamon, “The development and impact of 454 sequencing” , Nature Biotech. 26, 1117 (2008)
 454 sequencing

The four dNTPs are flowed sequentially

across the plate

Sequencing is monitored electronically as

flashes of light from the individual wells
when a nucleotide is incorporated

Many reactions can be carried out

in parallel

Rothberg and Leamon, “The development and impact of 454 sequencing” , Nature Biotech. 26, 1117 (2008)
 454 sequencing

Can sequence ~250 bases per well

Have 1,600,000 wells per plate
Can sequence up to 400,000,000 bases per plate (8 hrs)
First individual human genome sequence

454 sequencing was used for the first

individual human genome sequence
project (2008)

Cost < $1,000,000

Genome sequence: James Watson
Reversible terminator sequencing
 Reversible terminator sequencing

Basis for the Illumina DNA sequencing method

 Reversible terminators

Bentley et.al. “Accurate whole human genome sequencing using reversible terminator chemistry” Nature 456, 53 (2008)
 Reversible terminator chemistry

Add all four 3´-O-azidomethyl dNTPs,

each labeled with a different
fluorophore

DNA-OH polymerase Add DNA polymerase

Determine identity of incorporated

nucleotide by fluorescence
(only one nucleotide is added at a time)
tris(2-carboxyethyl)phosphine
(TCEP)
Remove fluorophore and 3´-O-azidomethyl
blocking group

linker scar
Regenerates a free 3´-hydroxyl group
for another cycle of nucleotide
addition

Bentley et.al. “Accurate whole human genome sequencing using reversible terminator chemistry” Nature 456, 53 (2008)
 Bridge amplification

DNA
fragment ligate PCR

Genomic DNA fragments are generated by random shearing

Fragments are ligated to a pair of forked adaptor oligonucleotides

Ligated products are amplified by PCR (using PCR primers that

are complementary to the adaptor sequences)
Forms double-stranded blunt-ended products with a different
adaptor sequence on each end
Bentley et.al. “Accurate whole human genome sequencing using reversible terminator chemistry” Nature 456, 53 (2008)
 Bridge amplification

3´ 3´ 3´ 3´
denature anneal

DNA fragments are denatured

Separated single strands are annealed to complementary PCR

primers that are covalently attached to a flow cell surface

Bentley et.al. “Accurate whole human genome sequencing using reversible terminator chemistry” Nature 456, 53 (2008)
 Bridge amplification

3´ 3´

3´ 3´ 3´ 3´
copy denature

A new strand is copied from the original strand in an extension

reaction (Bst I polymerase) that is primed from the 3´-end of
the surface-bound PCR primer

The original strand is then removed by denaturation; the copy

strand remains covalently attached to the surface-bound PCR
primer
Bentley et.al. “Accurate whole human genome sequencing using reversible terminator chemistry” Nature 456, 53 (2008)
 Bridge amplification

3´ 3´ 3´

repeat
3´
1. bridge denature
2. copy 3´ 3´

The adaptor sequence at the 3´-end of the copy strand can anneal to
a second surface-bound PCR primer
The bridging strand serves as a template for an extension reaction
that is primed from the 3´-end of the second PCR primer
Multiple cycles of annealing, extension, and denaturation result in
the growth of clonal DNA clusters

Bentley et.al. “Accurate whole human genome sequencing using reversible terminator chemistry” Nature 456, 53 (2008)
 Clonal single molecule arrays

Can generate ~1000 copies of the DNA segment per cluster

And can have 100,000,000 clusters per cm2

 Reversible terminator sequencing

3´ 3´
3´ 3´

3´
linearize denature 3´ sequence 3´
3´ 3´

The DNA in each cluster is linearized by cleavage within one adaptor

sequence and then denatured

Generates a unique single-stranded template which can then be

used for reversible terminator sequencing

Bentley et.al. “Accurate whole human genome sequencing using reversible terminator chemistry” Nature 456, 53 (2008)
Illumina DNA sequencing

Can only sequence ~35 bases per

reaction

But can monitor millions of reactions

simultaneously (in parallel)
 Illumina DNA sequencing

Illumina DNA sequencer 9° N polymerase

Reengineered the active site of 9° N DNA polymerase to improve the

efficiency of incorporation of the reversible terminator dNTPs
Ion Torrent sequencing
 DNA polymerase

H+
H+

Each nucleotide incorporation step is accompanied by

the release of a proton (H+)
 Ion Torrent sequencing

In both 454 and Ion Torrent sequencing:

DNA fragments are immobilized and amplified
on beads, which are then placed in the wells
of a chip
The four dNTPs are then flowed sequentially
over the wells of the chip

In 454: nucleotide incorporation is detected

by the release of PPi (which triggers a series of
enzymatic reactions that results in the emission
of light)
In Ion Torrent: nucleotide incorporation is
detected by the release of H+ (which results
in a decrease in the pH of the reaction solution
in the well)
Churko et. al. “Overview of high throughput sequencing technologies “
Circ. Res. 112, 1613 (2013)
 Ion Torrent DNA sequencing

Ion torrent DNA sequencer DNA ionogram

Ion Torrent website